a) Field of the Invention
The invention is directed to a process for the device configuration of confocal microscopes, preferably of laser scanning microscopes, in which laser light with one or more spectral lines is generated and directed on a specimen which contains a fluorescent dye or on which a fluorescent dye has been applied, wherein the light reflected and/or emitted by the specimen serves as basis for an image evaluation and the quality of the image evaluation is influenced in that filters or filter combinations which correspond to the emission wavelength of the fluorescent dye are introduced into the microscope beam path. The invention is further directed to an arrangement for carrying out this process.
b) Description of the Related Art
Confocal microscopy, as a special further development of light microscopy, offers the possibility not only of a higher resolution of microstructures, but also imaging and measurement of these microstructures in the Z-coordinate of space. As a result of this, fluorescence technique has increasingly become a central focus of interest in the field of precision instrument construction and in science apart from the conventional contrasting methods of bright field, phase contrast and interference contrast.
Fluorescence technique is based on the fact that different fluorochromes whose excitation wavelengths and emission wavelengths lie in different spectral bands make it possible to show structures in a plurality of colors simultaneously. Thus, depending on the spectral characteristics of different dye molecules, information concerning the physiological parameters can be obtained in addition to morphological information.
When the confocal microscope is used for fluorometric processes, information may be deduced concerning changes in the concentration of ions and molecules. Also significant are indicators which, in addition to intensity dependence, point to a shift in the excitation spectrum or emission spectrum and, in this way, enable a quantification of ion concentrations. Individual lasers, each with a wavelength, or a "multi-line" mixed gas laser with a plurality of usable wavelengths are used as illumination sources.
A procedure of this kind, including the associated instrument technology, is described in "Mitteilungen fur Wissenschaft und Technik [Science and technology notes]", Vol. II, No. 1, pages 9-19, June 1995. It is explained in detail therein that the design of the detection system must also be adapted to the emission wavelength in accordance with the steps carried out in the illumination path for point-accurate object illumination with different excitation wavelengths in the opposite direction. This entails detecting the spectrally different information from exactly the same region of the specimen, recording this information in exact pixels and preparing it for the image evaluation. Only in this way is it possible to record 3D data records which allow, for example, a reliable correlation of spatial cellular or tissue structures within the microarchitecture or the localization of a plurality of gene sites in chromosomes.
For sequential detection, for example, in the evaluation of reflections and emitted radiation in the fluorescent process, neutral splitters and single-dichroic splitter mirrors are used as excitation beam splitters in the confocal beam path, wherein it is necessary to exchange splitters between successive recordings. Blocking filters are used, for example, to limit the detected emission bands, wherein long-pass filters and bandpass filters can be used, if desired, for fine adjustment of the spectral separation.
All of the filter components are mounted in motor-operated filter wheels and are ready for use in that they can be exchanged for one another when correspondingly driven.
While the branching of the emission light into a plurality of detection channels has the advantage that illumination pinholes and detection pinholes are arranged in an exact confocal manner for all detectors, an increase in the number of detection channels results in an increase in the number of possible filter combinations. Consequently, a user of the confocal microscope must know the exact excitation wavelength and emission wavelength of a fluorescent dye in order to be able to make an image of the specimen or the image of a selected plane of the specimen with the microscope.
The invention utilizes, for example, the effect according to which the energy radiated into the dye with the excitation wavelength is subsequently transformed into a wavelength of lower energy and is radiated back from the dye. In this connection, the emission photons travel via the above-described mirrors, filters and color splitters in the respective detection channel where they encounter a photomultiplier (PMT) which detects and records them and makes them available for image processing.
In this connection, the path taken by every photon from the specimen to one of the detectors depends on the device setting that the user has selected. Thus, it is possible owing to subjective influences that the path predetermined for the photons by the selected device configuration can be correct, unfavorable or incorrect.
The setting of the device configuration is correct when laser light has been selected which has a spectral composition corresponding to the excitation radiation for a dye contained in the specimen and when the color splitters and filters whose transmission spectrum corresponds to the emission wavelength of the dye have been swung into the microscope beam path on the emission side.
A setting is unfavorable, i.e., functional but not optimum, when, for example, a laser configuration has been selected which corresponds to the excitation radiation of the dye, but the color splitters or filters which have been swung into the emission beam path only pass a portion of the emission spectrum. As a result, only weaker-than-optimum signals reach the respective detector. Selection of a laser wavelength which does not exactly correspond to the excitation wavelength of the selected dye has a similar effect.
A totally wrong setting results when the wrong laser is selected on the excitation side and/or a mirror combination or filter combination with a transmission spectrum adjacent to the emission spectrum has been selected on the emission side. The result is that the entire system cannot generate any images.